Methamphetamine (METH) is one of the most commonly abused drugs among HIV-infected individuals in the United States. METH use is associated with the worst health outcomes in HIV-infected individuals, as METH adversely impacts immunological responses to viral infections. Although METH has been indicated as a facilitating factor in the development of HIV disease, the mechanism(s) of METH actions on HIV remains to be determined. Our in vitro studies showed that METH could enhance HIV replication and impair intracellular innate immunity. Thus, we proposed further studies using the clinical specimens from the unique and well-characterized study subjects, investigating the in vivo and ex vivo impact of METH on RIG- I/TLR3 signaling-mediated anti-HIV activities. We hypothesize that METH suppresses intracellular innate immunity in primary immune cells (CD4+T cells, monocytes) and CNS cells (astrocytes, microglia), facilitating HIV infection and neuronal injury. We propose three specific aims to address this hypothesis: In aim 1, we will determine the plasma levels of exosome- derived HIV restriction miRNAs and immune activation markers in METH users with or without chronic HIV infection. In aim 2, we will determine the ex vivo and in vitro effects of METH on RIG-I/TLR3 signaling-mediated innate immunity against HIV. We will also examine the mechanisms involved in the actions of METH on RIG-I/TLR3 signaling and HIV infection. In aim 3, we will determine the effect of astroglial RIG-I/TLR3 signaling on HIV replication in microglia (glia-glia interaction) as well as the impact of METH on astroglial RIG-I/TLR3 signaling-mediated anti-HIV effect and neuronal cell protection. These proposed novel studies with combinational in vivo, ex vivo and in vitro approaches are clinically significant and relevant, as they will reveal previously unidentified mechanisms for the METH actions on HIV and the CNS. In addition, the proposed studies will identify the novel immunological markers for monitoring the impact of METH use on the development of HIV disease.